Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Summary

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Abstract

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH₂). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O₂ min^-1 mg total mitochondrial protein^-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Introduction

The history of plant mitochondrial research goes back over 100 years¹. Intact mitochondria were first isolated in the early 1950s using differential centrifugation. The advent of a colloidal density gradient in the 1980s allowed mitochondria to be purified without suffering osmotic adjustment. While gradient purified mitochondria are suitable for most purposes, due to the sensitivity of mass spectrometry, even relatively minor contaminants can be detected and may be inappropriately assigned a mitochondrial location². The use of free flow electrophoresis can remove both plastidic and peroxisome contamination³, but free flow electrophoresis is a highly specialized technique and is not required for the vast majority of studies. Furthermore, when determining the location of a protein it needs to be remembered that dual or multiple targeting of proteins occurs in cells. Over 100 dual targeted proteins are described for chloroplasts/plastids and mitochondria⁴, and a number of proteins targeted to mitochondria and peroxisomes are also known⁵. Furthermore, the re-location of proteins under specific stimuli, e.g. oxidative stress, is an emerging theme in cell biology⁶. Thus, the location of proteins needs to be considered in the context of the biology studied, and a variety of approaches are used to determine and verify location².

Mitochondria are typically isolated from plant tissues by homogenization, a balance is required between breaking open the cell wall to release mitochondria, and not damaging the mitochondria. Traditionally, with potato and cauliflower, homogenization involves using household blender/juicer apparatus to make a liquid extract in a buffer with various components to maintain activity. Isolation of mitochondria from pea leaves, (a popular material for mitochondrial isolation using young seedlings (~10 days old), utilizes a blender to lyse cells as the leaf material is soft. With the availability of Arabidopsis thaliana T-DNA insertional knock-out lines, the need to be able to purify mitochondria to carry out functional studies has necessitated the development of methods to isolate mitochondria from leaf, root or flower tissue. Overall the methods developed for other plants worked well⁷, with the perquisite that grinding of the material needed to be optimized. For Arabidopsis this can be achieved in a variety of ways (see below), and differs between tissue types (root versus shoot). The use of the continuous gradient can also be optimized as the density of mitochondria from different organs or developmental stages means they can migrate differently. Thus, for maximum separation the density of the gradient can be refined to ensure to achieve best separation.

Once purified the mitochondria can be used for a variety of studies, including protein and tRNA uptake experiments, enzyme activity assays, respiratory chain measurements and western blot analyses. Isolated mitochondria can also be used for mass spectrometry analyses of protein abundance. Targeted multiple reaction monitoring (MRM) analyses allows for the quantification of defined proteins, but require significant assay development. In contrast, quantification by dimethyl or other isotope labels⁸, provides a discovery approach in identifying differences across the whole proteome that can be used to uncover novel biological insights.

Protocol

This protocol is used for the isolation of intact mitochondria from Arabidopsis thaliana organs grown on soil using continuous colloidal density gradients. All procedures following the collection of the material are carried out at 4 °C.

1. Preparation of Grinding Medium, Wash Buffer, and Gradient Solutions

1. Prepare 300 mL of grinding medium (minus ascorbate and cysteine) and 200 mL of 2x wash buffer per Table 1 one day prior to the isolation, keep them cold at 4 °C.
   NOTE: Add sodium ascorbate and L-cysteine (free base) to a final concentration of 17.84 mM and 20.36 mM, respectively, to the grinding medium on the morning of the isolation. If the mitochondria are to be used for western blot or blue native-polyacrylamide gel electrophoresis (BN-PAGE) analyses, make up 2x wash buffer without BSA.
2. Prepare gradients on the morning of mitochondrial isolation (Figure 1 and Table 1).
   1. Make the heavy and light gradient solutions in two beakers; 35 mL of each is sufficient for two gradient tubes.
   2. Wash the chambers of the gradient pourer and PVC peristaltic tubing with deionized water and ensure even flow through the tubing from all tubing.
   3. Place 2 centrifuge tubes (50 mL) at a slight angle on ice with the PVC peristaltic tubing outlets taped to the inside of the tubes to ensure the gradient solution runs down the side of the tubes.
   4. Close the connection between the inner and outer chambers (black lever down). Place gradient pourer on a magnetic stirrer with a small stir bar in the inner chamber.
   5. Pour the 35 mL heavy gradient solution into the inner chamber (chamber with tubing outlet). Dispense the heavy gradient solution into the two centrifuge tubes placed on ice until half is remaining with the peristaltic pump rate set to fast (300 mL/h).
   6. Pour the 35 mL light gradient solution into the outer chamber (chamber without tubing outlet). Open the connection between the chambers (push black lever up halfway) and allow solutions to mix gently. Mix the solutions using the magnetic stirrer.
   7. Allow the solutions to run slowly (60 mL/h) until all of the gradient mix has been dispensed from the chambers resulting in two 0 - 4.4 % (w/v) polyvinylpyrrolidone (PVP) gradient-filled tubes. Keep the gradients on ice until ready to use in Step 2.12.
      NOTE: Once the gradient has been prepared, dilute 2x wash medium to 1x with prechilled-deionized water and keep cold at 4 °C.

2. Homogenization and Mitochondrial Isolation

1. Cut whole rosette tissue from 4-week old Arabidopsis plants grown on soil with scissors, at least 80 plants will be required for 1 prep.
   NOTE: 10 - 14 day old water cultures, minimum 5 pots/prep, or 2-week old seedlings grown on Murashige & Skoog Basal Salt Mixture (MS) agar plates, minimum of 4 plates, may also be used.
2. Place half of the plant material that has been cut into small pieces in a pre-cooled 4 °C large mortar and pestle with 75 mL of grinding medium (Table 1), and grind extensively for several minutes until no big pieces of tissue are left.
3. Pre-wet 4 layers of filtration material (22 - 25 µm pore size) with grinding medium and filter homogenate through the 4 layers of filtration material through a plastic funnel into a 500 mL conical flask.
4. Grind the remaining half of the tissue with another 75 mL of grinding medium.
5. Combine homogenates in the filtration materials and filter as much as possible homogenate through.
6. Grind the remaining tissue remaining on the filtration material again with the remaining 150 mL of grinding medium, filter again into the 500 mL conical flask.
7. Centrifuge the filtered homogenate in 8 pre-chilled 50 mL plastic centrifuge tubes at 2,500 x g at 4 °C in a fixed angle rotor for 5 min.
8. Pour the supernatant into clean centrifuge tubes (50 mL; avoid disturbing the green pellet) and centrifuge at 17,500 x g at 4 °C for 20 min in a fixed angle rotor.
9. Discard all but <3 mL of the supernatant by aspiration (or gently pour off without disturbing the pellet). Gently resuspend the pellet in the residual supernatant using a small fine paintbrush.
10. Add 10 mL of 1x wash buffer to each tube and combine 4 tubes into 1 clean centrifuge tube (i.e., resulting in 2 tubes).
11. Fill these tubes with 1x wash buffer to 50 mL and repeat steps 2.7 - 2.9.
12. Pool crude mitochondrial pellets into 1 tube using a dropper and disperse evenly with the fine paintbrush (a small amount of wash buffer may be used but the final volume needs to be less than 3 mL to fit on top of the gradient). Gently layer the mitochondrial suspension with a dropper onto the 2 continuous 0 - 4.4 % (w/v) PVP gradients.
13. Balance tubes by weight (using 1x wash buffer) and centrifuge at 40,000 x g at 4 °C for 40 min. Ensure the break is turned off. Mitochondria will migrate to a band near the bottom of the tube, or may be present in the heavy solution at the bottom of the tube (identified by a cloudy, yellowish band; Figure 2).
14. Carefully remove the upper 5 cm of solution above the mitochondrial band by aspiration.
15. Distribute the remaining solution containing the mitochondria into 2 clean tubes and fill with 1x wash buffer. Evenly distribute the colloidal density gradient and mitochondria by covering the mouth of the tube with a plastic paraffin film and inverting several times. Centrifuge for 15 min at 31,000 x g at 4 °C with slow braking.
    NOTE: Before centrifugation, make sure the mitochondria and the remaining colloidal density gradient are evenly distributed. Otherwise, the colloidal density gradient may concentrate at the bottom of the tube and prevent pelleting of the mitochondria.
16. Remove the supernatant by aspiration, fill the tube with 1x wash buffer up to 50 mL, and invert again to ensure mixing. Centrifuge for 15 min at 31,000 x g at 4 °C with slow braking.
17. Aspirate the supernatant and collect the mitochondrial pellets in as small a volume (~500 µL) as possible using a pipette with modified 200 µL tips (cut the tip a little above its end to increase its opening). Place the mitochondria into a 1.5 mL tube and keep on ice for immediate use or store at -80 °C.
    NOTE: If mitochondria will be used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), BN-PAGE, or immunoblot analyses, use 1x wash buffer without BSA for the final washes (i.e., step 2.15 and 2.16).
18. Determine the protein concentration of mitochondria using the Bradford method.
    NOTE: For BN-PAGE centrifuge aliquots at 4 °C, and store pellet at -80 °C until use. For oxygen consumption measurements, only freshly isolated mitochondria can be used.

3. Oxygen Consumption Measurements

NOTE: Oxygen consumption by freshly isolated plant mitochondria can be analyzed with a Clark-type oxygen electrode, enabling the determination of mitochondrial intactness, cytochrome c pathway activity, and alternative pathway activity.

1. Software installation
   1. Install licensed oxygen monitoring software onto the computer used for oxygen consumption measurements.
2. Preparation of respiration medium, substrates, chemical stock solutions, and the Clark-type oxygen electrode
   1. Prepare respiration medium (300 mM sucrose, 10 mM NaCl, 5 mM KH₂PO₄, 2 mM MgSO₄, 0.1 % (w/v) bovine serum albumin (BSA), 10 mM TES (pH 7.2)) used for oxygen consumption assays.
   2. Prepare substrates, inhibitors, and effectors used for measuring mitochondrial respiration (Table 2). These can be prepared earlier and stored at -20 °C.
      NOTE: Neutralize the pH of acidic solutions such as organic acids to pH 7.0 using NaOH before use.
   3. Warm the respiration medium to the same temperature as the assay chamber (25 °C).
   4. Assemble the Clark-type oxygen electrode as described in the protocol of the manufacturer.
   5. Place 1 mL air-saturated water (air-saturated water is obtained by vigorously shaking a small quantity of deionized water in a large conical flask) in the measuring chamber. To switch on the stirrer, click the "Stirrer Speed" button. Click the "On" button and set the stirrer speed to 65 rpm. Click "OK" to continue. Stir the water continuously at 25 °C.
3. Calibration of oxygen electrode
   1. Warm the water that will be used for calibration to the same temperature as the assay chamber (25 °C).
   2. Wait for the current to stabilize and calibrate the oxygen electrode.
   3. To start calibration of the oxygen electrode, click the "Calibrate" button, select "Liquid Phase Calibration," and then "Air Saturated Water."
   4. A new window will open (Oxygen Calibration (Liquid Phase) - Step 1 of 5). Select the channel to calibrate (the channel the electrode control box is attached to) and enter variables (set "Chamber Temperature" to 25 °C and "Atmospheric Pressure" to 101.32 kPa). Click "OK "to continue.
   5. In the next step (Step 2 of 5), setup the stirrer speed (65 rpm) and click "OK" to continue.
   6. In step 3 of 5, wait for the signal to reach a plateau. The term "Plateau reached, press OK to continue" will appear in the box. Click "OK" to continue.
   7. In step 4 of 5, establish zero oxygen in the chamber by adding 5 mg sodium dithionite. Click "OK" to continue.
      NOTE: A decline in oxygen concentration should be visible in the trace and should reach 0.
   8. In the last step of calibration (Step 5 of 5), wait for the signal to reach a plateau. The term "Plateau reached, press OK to continue" will appear in the box. Click "OK" to continue.
   9. Click "Save Calibration" to save the calibration. A new window will open. Click "OK" to confirm to save the new calibration for the oxygen control box.
      NOTE: After successful calibration, the labeling "Oxygen (nmol/mL)" will appear on the y-axis.
   10. After calibration, clean the measuring chamber by rinsing the chamber with water (at least 5 - 10 times) to ensure proper cleaning. Place 1 mL of respiration medium into the measuring chamber and allow the membrane to equilibrate for a few minutes until the oxygen consumption trace is linear.
   11. Adapt the scale of the oxygen consumption trace to get a good slope view. Click "Zoom XY" and enter variables (set "Oxygen" to 0 - 300 and the "Time Axis" to 0 - 45 min).
4. Determination of mitochondrial integrity
   1. With plunger open, add 970 µL air-saturated respiration medium to the reaction chamber.
   2. Add 30 µL isolated mitochondria or 150 µg of total mitochondrial protein as determined after mitochondria isolation (total protein needs to be included in calculations afterwards) to the reaction chamber using a pipette with a cut tip.
   3. Stir the mitochondrial suspension continuously using a magnetic stirrer bar (65 rpm) at 25 °C to air-saturate the solution.
   4. Seal the chamber with the plunger, click "Start Recording" to record oxygen consumption, and allow the oxygen consumption trace to stabilize (1 - 2 min).
   5. Determine the rate of oxygen consumption manually after adding 10 mM ascorbate and 25 µM cytochrome c for 5 min to get the "before detergent" rate.
      NOTE: To label the addition of substrates, inhibitors, and effectors directly in the trace, click the "Add Event Mark" button and enter the corresponding label designation. Click "OK" to continue.
   6. Determine the rate of oxygen consumption manually for 3 min after adding 0.05% (v/v) detergent to give the "after detergent" rate. Click "Stop Recording."
   7. Click the "Rates of Change Table" button. 2 cursors will appear as vertical lines on the graph screen. Click and drag the pair of rate cursors to the required positions in the trace to define the rate interval. Move the rate interval along the trace using the left rate cursor. Set the rate interval itself using the right rate cursor. The oxygen monitoring software automatically draws a line of best fit between the two cursors while moving the rate interval cursors along the trace.
   8. Once the desired rate interval and position has been defined, enter the rate into the table with a right click on 1 of the 2 cursors, and select "Add Rate to Table." Click the "Add" button to confirm to display the rate on the main rate table.
   9. Calculate mitochondrial integrity by dividing the "before detergent" rate by the "after detergent" rate, expressed as a percentage, and subtract it from 100.
5. Determination of mitochondrial respiration
   1. With plunger open, add 970 µL of air-saturated respiration medium to the reaction chamber. Add 500 µM ATP and 30 µL isolated mitochondria or 150 µg of total mitochondrial protein using a pipette with a cut tip to the respiration medium in the reaction chamber.
   2. Stir the mitochondrial suspension continuously using a magnetic stirrer bar (65 rpm) at 25 °C.
   3. Close plunger, click "Start Recording" to record oxygen consumption, and wait for 1 - 2 min for oxygen consumption rate to stabilize (when the oxygen consumption trace is linear). Add 5 mM succinate. Record the rate of oxygen consumption for about 2 min.
      NOTE: To label the addition of substrates, inhibitors, and effectors directly in the trace, click the "Add Event Mark" button and enter the corresponding label designation. Click "OK" to continue.
   4. Add 1 mM adenosine diphosphate (ADP). Continue recording the rate of oxygen consumption for 2 min.
   5. Add 1 mM NADH. Record the rate of oxygen consumption for 4 - 8 min. This rate is the capacity of respiration via cytochrome oxidase.
   6. Add 1 mM potassium cyanide (KCN) (or 2.5 µM myxothiazol or 5 µM antimycin A) to inhibit the cytochrome c pathway, and continue to record for about 2 min to observe oxygen consumption via the alternative oxidase.
   7. Add 5 mM dithiothreitol (DTT) and 10 mM pyruvate to fully activate the alternative oxidase. Continue to record for 5 - 7 min: This is the capacity of the alternative oxidase.
   8. Add 500 µM n-propyl gallate (nPG), and record for 2 min. This assesses residual oxygen consumption rate that cannot be chemically inhibited. Subtract this rate from the other rates recorded, as it is not likely to be mitochondrial in origin. Oxygen consumption still occurring after inhibition of the cytochrome c and AOX pathway (by KCN and nPG) is very likely to come from peroxidases present as contaminants in the isolated mitochondria⁹.
   9. Click "Stop Recording."
   10. Click the "Rates of Change Table" button and enter the rate into the table with a right click on one of the two cursors, and select "Add Rate to Table". Click the "Add" button to confirm to display the rate on the main rate table.
       NOTE: When adding effectors dissolved in organic solvents (e.g., antimycin A, nPG, myxothiazol), the chamber and plunger must be rinsed with ethanol and then water to ensure proper cleaning.

Results

Using this protocol, we were able to detect different mitochondrial proteins by SDS-PAGE and immunoblotting. As shown in Figure 3A, the protein isolated from water culture tissue is sufficient to detect a faint band (2 µg). Signal intensity increases proportionately to the amounts loaded. For mitochondria isolated from tissues grown on plates (Figure 3B), the response to high light stress treatment in different genotypes can be a...

Discussion

Typically, isolation of mitochondria from Arabidopsis leaves yields up 3 mg of mitochondria from approximately 80 - 100 3 - 4-week old plants, although yields of greater than 5 mg can often be achieved with thorough grinding. The yield varies with growth conditions and decreases dramatically as leaves senesce, although mitochondria structure seems to be well maintained during senescence⁹. One of the most critical features to obtain a good yield is the method of grinding to lyse cells to release mi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ADP	Sigma-Aldrich	A2754	Chemical
Antimycin A	Sigma-Aldrich	A8674	Chemical, dissolve in ethanol
AOX antibody	from Tom Elthon		Elthon et al., 1989
Ascorbate	Sigma-Aldrich	A0157	Ascorbate Oxidase from Cucurbita sp.
ATP	Sigma-Aldrich	A26209	Chemical
Bovine serum albumin (BSA)	Bovogen	BSAS 1.0	Chemical
Clarity western ECL substrate	Bio-Rad Laboratories	1705061	Chemical
Criterion Stain-Free Precast Gels 8-16% 18 Wells	Bio-Rad Laboratories	5678104	Chemical
Cyanide	Sigma-Aldrich	60178	Chemical
Cytochrome c	Sigma-Aldrich	C3131	Chemical
Difco Agar, granulated	BD Biosciences	214530	Chemical
Dithiotreitol	Sigma-Aldrich	D0632	Chemical
EDTA disodium salt	Sigma-Aldrich	E5134	Chemical
Gamborg B-5 Basal Medium	Austratec	G398-100L	Chemical
Gamborg Vitamin Solution (1000x)	Austratec	G219-100ML	Chemical
Goat Anti-Mouse IgG (H + L)-HRP Conjugate	Bio-Rad Laboratories	1706516-2ml	Chemical
Goat Anti-Rabbit IgG (H + L)-HRP Conjugate	Bio-Rad Laboratories	1706515-2ml	Chemical
L-Cysteine	Sigma	C7352-100G	Chemical
Magnesium sulfate	Sigma-Aldrich	230391	Chemical
Murashige & Skoog Basal Salt Mixture (MS)	Austratec	M524-100L	Chemical
Myxothiazol	Sigma-Aldrich	T5580	Chemical, dissolve in ethanol
NADH	Sigma-Aldrich	N8129	Chemical
Ndufs4 antibody	from Etienne Meyer		Meyer et al., 2009
n-Propyl gallate	Sigma-Aldrich	P3130	Chemical, dissolve in ethanol
Percoll	GE Healthcare	17-0891-01	Chemical, colloidal density gradient
Polyvinylpyrrolidone (PVP40)	Sigma-Aldrich	PVP40	Chemical
Potassium cyanide	Sigma-Aldrich	60178	Chemical
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P5655	Chemical
Pyruvate	Sigma-Aldrich	P2256	Chemical
Sodium chloride	Chem-Supply	SA046	Chemical
Sodium dithionite	Sigma-Aldrich	157953	Chemical
Sodium L-ascorbate	Sigma	A4034-100G	Chemical
Succinate	Sigma-Aldrich	S2378	Chemical
Sucrose	Chem-Supply	SA030	Chemical
TES	Sigma-Aldrich	T1375	Chemical
Tetrasodium pyrophosphate (Na4P2O7 · 10H2O)	Sigma-Aldrich	221368	Chemical
Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit	Bio-Rad Laboratories	1704271	Chemical
Triton-X 100	Sigma-Aldrich	X100	Chemical, detergent
Western Blocking Reagent	Sigma	11921681001	Chemical
Balance	Mettler Toledo	XS204	Equipment
Beakers	Isolab	50 mL
Centrifuge	Beckman Coulter	Avanti J-26XP	Equipment
Centrifuge tubes	Nalgene	3117-9500	Equipment
Circulator	Julabo	1124971	Attached to oxygen electrode chamber
Conical flask	Isolab	500 mL
Dropper		3 mL
Fixed angle rotor	Beckman Coulter	JA25.5	Equipment
Funnel	Per Alimenti	14 cm	For filtering
Gradient pourer	Bio-Rad	165-4120	For preparation of gradients
Magnetic Stirrer ATE	VELP Scientifica	F20300165	Equipment
Miracloth	VWR	EM475855-1R	Filtration material
Mortar and pestle	Jamie Oliver	Granite, 6 Inch	Equipment
O2view	Hansatech Instruments		Oxygen monitoring software
Oxygraph Plus System	Hansatech Instruments	1187253	Clark-type oxygen electrode
Paintbrush	Artist first choice	1008R-12
Parafilm	Bemis	PM-996	plastic paraffin film
Peristaltic pump	Gilson	F155001	For preparation of gradients
PVC peristaltic tubing	Gilson	F117930	For preparation of gradients
Water bath	VELP Scientifica	OCB	Equipment